Regulation Of Defense Responses Research Articles

Unraveling the role of nitric oxide in regulation of defense responses in chilli against Alternaria leaf spot disease

Alternaria leaf spot disease of chilli is an emerging problem in different chilli growing regions of the world including India. To understand the biochemical and molecular backbone of defense response against Alternaria alternata (A. alternata), two chilli cultivars (one tolerant and one susceptible) were selected. Different biochemical and histochemical methods were employed to understand the differential defense responses of two chilli cultivars in response to Alternaria invasion. Semiquantitative RT-PCR analysis showed higher transcript accumulation of different defense related genes in tolerant cultivar. The nitric oxide (NO) status was also monitored during activation of defense responses. In response to this fungus, reactive oxygen species (ROS) generation, callose deposition, cell death was also checked in both the cultivars. The tolerant cultivar showed enhanced defense responses (including NO) in response to the fungus, than that of susceptible cultivar. However, the tolerant cultivar showed reduced disease symptoms, lower amount of lipid peroxidation, lower amount of hydrogen peroxide (H2O2) generation and lower amount of cell death. In NO depleted condition pathogen induced defense responses were significantly compromised in both the cultivars. Overall, our present study provides a glimpse of NO participation in activation of defense responses in chilli against Alternaria invasion.

EgJUB1 and EgERF113 transcription factors as potential master regulators of defense response in Elaeis guineensis against the hemibiotrophic Ganoderma boninense

BackgroundHemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection.ResultsThe present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells.ConclusionOur findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

Magnaporthe oryzae systemic defense trigger 1 (MoSDT1)-mediated metabolites regulate defense response in Rice

BackgroundSome of the pathogenic effector proteins play an active role in stimulating the plant defense system to strengthen plant resistance.ResultsIn this study, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) was implemented to identify altered metabolites in transgenic rice containing over-expressed M. oryzae Systemic Defense Trigger 1 (MoSDT1) that was infected at three-time points. The characterized dominating metabolites were organic acids and their derivatives, organic oxygen compounds, lipids, and lipid-like molecules. Among the identified metabolites, shikimate, galactinol, trehalose, D-mannose, linolenic acid, dopamine, tyramine, and L-glutamine are precursors for the synthesis of many secondary defense metabolites Carbohydrate, as well as amino acid metabolic, pathways were revealed to be involved in plant defense responses and resistance strengthening.ConclusionThe increasing salicylic acid (SA) and jasmonic acid (JA) content enhanced interactions between JA synthesis/signaling gene, SA synthesis/receptor gene, raffinose/fructose/sucrose synthase gene, and cell wall-related genes all contribute to defense response in rice. The symptoms of rice after M. oryzae infection were significantly alleviated when treated with six identified metabolites, i.e., galactol, tyramine, L-glutamine, L-tryptophan, α-terpinene, and dopamine for 72 h exogenously. Therefore, these metabolites could be utilized as an optimal metabolic marker for M. oryzae defense.

Changes in the expression of CrFTA, the Catharanthus roseus farnesyltransferase α-subunit gene, in response to a Candidatus Liberibacter asiaticus infection

The farnesyltransferase α-subunit (FTA) may be involved in the regulation of defence responses against pathogens in plants. In this study, this gene was amplified from Catharanthus roseus (CrFTA gene). The cDNA was found to be 1 403 bp long, and encodes a putative protein of 331 amino acids that contains a conserved PPTA motif. The phylogenetic analysis showed that the sequence of CrFTA is the most similar to that from Coffea canephora. The qRT-PCR assays indicated that CrFTA is expressed in the leaves, stems, and roots. During a Candidatus Liberibacter asiaticus (Ca. L. asiaticus) infection, the CrFTA expression levels significantly increased and reached 18-fold that measured in the control group, after which its expression decreased gradually from 22 days after top-grafting (DAT) to the end of the experiment. Spray application of Manumycin A (ManuA), a specific inhibitor of farnesyltransferase, on the leaves of C. roseus plants caused a significant decrease in the CrFTA expression and a significant increase in the Ca. L. asiaticus positivity percentage after top-grafting with the Ca. L. asiaticus-infected shoots compared with the groups not treated with ManuA. Furthermore, ABA had no significant effect on the relative expression of CrFTA and the number of Ca. L. asiaticus-positive plants. These results suggest that CrFTA most likely plays a role in mediating the tolerance to a Ca. L. asiaticus infection in C. roseus.

FaWRKY11 transcription factor positively regulates resistance to Botrytis cinerea in strawberry fruit

Plant WRKY transcriptional factors play an important role in responses to biotic and abiotic stresses and are involved in a wide range of resistance strategies against pathogen attacks. The present study investigated the functional role of FaWRKY11 in the regulation of defense responses to B. cinerea in strawberry fruit (Fragaria ananassa Duch. cv. Benihoppe). As strawberry fruits ripened and developed, FaWRKY11 expression levels gradually decreased and were closely related to the severity of B. cinerea. Transient transformation was conducted to overexpress and silence FaWRKY11 in strawberry fruits, which were later analyzed upon B. cinerea inoculation. The results indicated that 5 days following infestation with B. cinerea, the tissue damage of the fruits with overexpressed FaWRKY11 was significantly alleviated. The disease incidence was significantly lower that of the control. Conversely, the degree of damage suffered by the silenced fruit tissue increased to a certain extent, and the disease incidence was slightly higher than that of the control. The results revealed the positive regulatory function of FaWRKY11 in the resistance of strawberries against B. cinerea. Once the FaWRKY11 gene was overexpressed and silenced, the expression levels of the genes related to B. cinerea of the fruit were regulated to diverse extents. The most significant variations were observed in the expression levels of transcription factors related to disease resistance, and also in genes related to metabolism of phytohormones. Our study inferred that the FaWRKY11 might regulate the resistance of strawberries against B. cinerea through regulating some disease-resistance transcription factors and metabolic genes. Our study demonstrated the regulatory mechanism of FaWRKY11 regarding the defense in strawberries against B. cinerea.

Transcriptome analysis reveals that exogenous ethylene activates immune and defense responses in a high late blight resistant potato genotype

Ethylene (ET) is one of the many important signaling hormones that functions in regulating defense responses in plants. Gene expression profiling was conducted under exogenous ET application in the high late blight resistant potato genotype SD20 and the specific transcriptional responses to exogenous ET in SD20 were revealed. Analysis of differentially expressed genes (DEGs) generated a total of 1226 ET-specific DEGs, among which transcription factors, kinases, defense enzymes and disease resistance-related genes were significantly differentially expressed. GO enrichment and KEGG metabolic pathway analysis also revealed that numerous defense regulation-related genes and defense pathways were significantly enriched. These results were consistent with the interaction of SD20 and Phytophthora infestans in our previous study, indicating that exogenous ET stimulated the defense response and initiated a similar defense pathway compared to pathogen infection in SD20. Moreover, multiple signaling pathways including ET, salicylic acid, jasmonic acid, abscisic acid, auxin, cytokinin and gibberellin were involved in the response to exogenous ET, which indicates that many plant hormones work together to form a complex network to resist external stimuli in SD20. ET-induced gene expression profiling provides insights into the ET signaling transduction pathway and its potential mechanisms in disease defense systems in potato.

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.

The G-protein α subunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation

Verticillium wilt, a devastating disease in cotton caused by Verticillium dahliae, reduces cotton quality and yield. Heterotrimeric GTP-binding proteins, consisting of Gα, Gβ, and Gγ subunits, transducers of receptor signaling, function in a wide range of biological events. However, the function of Gα proteins in the regulation of defense responses in plants is largely unexplored, except for a few reports on model species. In the present study, a cotton G-protein α-subunit-encoding gene (GhGPA) was isolated from Verticillium wilt-resistant Gossypium hirsutum (upland cotton) cv. ND601. GhGPA transcription was up-regulated under V. dahliae stress, with higher expression in tolerant than in susceptible cotton cultivars. Subcellular localization revealed GhGPA to be located in the plasma membrane. GhGPA shows high (85.0%) identity with Arabidopsis AT2G26300 (AtGPA1), and AtGPA1 gpa1-4 mutants displayed susceptibility to V. dahliae. Ectopic expression of GhGPA successfully restored the resistance of Arabidopsis gpa1-4 mutants to Verticillium wilt and made them more resistant than the wild type. Overexpression of GhGPA in Arabidopsis markedly increased the resistance and resulted in dramatic up-regulation of pathogenesis-related (PR) genes and increased in H2O2 accumulation and salicylic acid (SA) and jasmonic acid (JA) contents. However, suppressing GhGPA expression via virus-induced gene silencing (VIGS) increased susceptibility to Verticillium wilt, down-regulated the expression of PR and marker genes in SA and JA signaling pathways, and reduced H2O2 content. The contents of SA and JA in Arabidopsis gpa1-4 and VIGS cotton were lower than those in the wild type and empty-vector control. However, GhGPA-overexpressing Arabidopsis contained more SA and JA than the wild type when inoculated with V. dahliae. Thus, GhGPA plays a vital role in Verticillium wilt resistance by inducing SA and JA signaling pathways and regulating the production of reactive oxygen species. These findings not only broaden our knowledge about the biological role of GhGPA, but also shed light on the defense mechanisms involving GhGPA against V. dahliae in cotton.

Identifying Breast Cancer-induced Gene Perturbations and its Application in Guiding Drug Repurposing

Background:Breast cancer is a complex disease with high prevalence in women, the molecular mechanisms of which are still unclear at present. Most transcriptomic studies on breast cancer focus on differential expression of each gene between tumor and the adjacent normal tissues, while the other perturbations induced by breast cancer including the gene regulation variations, the changes of gene modules and the pathways, which might be critical to the diagnosis, treatment and prognosis of breast cancer are more or less ignored.Objective:We presented a complete process to study breast cancer from multiple perspectives, including differential expression analysis, constructing gene co-expression networks, modular differential connectivity analysis, differential gene connectivity analysis, gene function enrichment analysis key driver analysis. In addition, we prioritized the related anti-cancer drugs based on enrichment analysis between differential expression genes and drug perturbation signatures.Methods:The RNA expression profiles of 1109 breast cancer tissue and 113 non-tumor tissues were downloaded from The Cancer Genome Atlas (TCGA) database. Differential expression of RNAs was identified using the “DESeq2” bioconductor package in R, and gene co-expression networks were constructed using the weighted gene co-expression network analysis (WGCNA). To compare the module changes and gene co-expression variations between tumor and the adjacent normal tissues, modular differential connectivity (MDC) analysis and differential gene connectivity analysis (DGCA) were performed.Results:Top differential genes like MMP11 and COL10A1 were known to be associated with breast cancer. And we found 23 modules in the tumor network had significantly different co-expression patterns. The top differential modules were enriched in Goterms related to breast cancer like MHC protein complex, leukocyte activation, regulation of defense response and so on. In addition, key genes like UBE2T driving the top differential modules were significantly correlated with the patients’ survival. Finally, we predicted some potential breast cancer drugs, such as Eribulin, Taxane, Cisplatin and Oxaliplatin.Conclusion:As an indication, this framework might be useful in understanding the molecular pathogenesis of diseases like breast cancer and inferring useful drugs for personalized medication.

WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana.

NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors. Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.

Gaining Insight into Exclusive and Common Transcriptomic Features Linked to Drought and Salinity Responses across Fruit Tree Crops.

The present study aimed at identifying and mapping key genes expressed in root tissues involved in drought and salinity tolerance/resistance conserved among different fruit tree species. Twenty-six RNA-Seq samples were analyzed from six published studies in five plant species (Olea europaea, Vitis riparia Michx, Prunus mahaleb, Prunus persica, Phoenix dactylifera). This meta-analysis used a bioinformatic pipeline identifying 750 genes that were commonly modulated in three salinity studies and 683 genes that were commonly regulated among three drought studies, implying their conserved role in resistance/tolerance/response to these environmental stresses. A comparison was done on the genes that were in common among both salinity and drought resulted in 82 genes, of which 39 were commonly regulated with the same trend of expression (23 were upregulated and 16 were downregulated). Gene set enrichment and pathway analysis pointed out that pathways encoding regulation of defense response, drug transmembrane transport, and metal ion binding are general key molecular responses to these two abiotic stress responses. Furthermore, hormonal molecular crosstalk plays an essential role in the fine-tuning of plant responses to drought and salinity. Drought and salinity induced a different molecular “hormonal fingerprint”. Dehydration stress specifically enhanced multiple genes responsive to abscisic acid, gibberellin, brassinosteroids, and the ethylene-activated signaling pathway. Salt stress mostly repressed genes encoding for key enzymes in signaling proteins in auxin-, gibberellin-(gibberellin 2 oxidase 8), and abscisic acid-related pathways (aldehyde oxidase 4, abscisic acid-responsive element-binding protein 3). Abiotic stress-related genes were mapped into the chromosome to identify molecular markers usable for the improvement of these complex quantitative traits. This meta-analysis identified genes that serve as potential targets to develop cultivars with enhanced drought and salinity resistance and/or tolerance across different fruit tree crops in a biotechnological sustainable way.

Structural insights into Arabidopsis ethylene response factor 96 with an extended N-terminal binding to GCC box

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.

Isolation and functional characterization of the mitogen-activated protein kinase kinase gene MAPKK1 from Panax notoginseng

Mitogen-activated protein kinase (MAPK) cascades are important signal transduction mechanisms that connect plant cellular and nuclear responses, which play key roles in plant development and stress responses. Here, the novel MAPK kinase (MAPKK) gene MAPKK1 was isolated from Panax notoginseng (Burk) F.H. Chen. Exogenous methyl jasmonate (MeJA), salicylic acid (SA), ethylene, and hydrogen peroxide treatments induced the transcription level of PnMAPKK1 in P. notoginseng roots. Additionally, PnMAPKK1 expression actively responded to Fusarium solani infection, a major causal agent of P. notoginseng root rot disease. The PnMAPKK1 gene was further fused with the green fluorescent protein gene in a plant expression vector and transformed into onion (Allium cepa) epidermal cells. Laser scanning confocal microscopy confirmed that the PnMAPKK1 protein localized in the cytoplasm. In addition, the plant overexpression vector pCAMBIA2300s-PnMAPKK1 was constructed and transformed into tobacco (Nicotiana tabacum L. cv. Xanthi). The PnMAPKK1 transgenic tobacco lines showed much stronger resistance levels to F. solani infection than the wild type. Moreover, the JA and SA contents in the PnMAPKK1 transgenic tobacco lines were significantly higher than those in wild type during F. solani infection. The up-regulation of several marker genes (PAL4, PR1c, WRKY1, Defensin, and LOC4) involved in JA and SA signaling was correlated with the overexpression of PnMAPKK1 in tobacco. Thus, the PnMAPKK1 gene is a positive regulator of defense responses to F. solani in P. notoginseng.

Gene Profiling of Compatible and Incompatible Interactions between Potato and Phytophthora infestans

Potato late blight, caused by the oomycete pathogen, Phytophthora infestans de Bary, is one of the most devastating plant diseases in the world. It is of great importance to screen disease resistance germplasms and elucidate the molecular mechanisms underlying the resistance. In a previous study, Huasong 7, a tetraploid potato variety, was identified to possess resistance to multiple P. infestans races at various levels. In the present study, Huasong 7 was inoculated with an incompatible isolate 428–2 and a compatible P. infestans isolate T30–4, and gene expression was profiled at 0, 24, 48, and 72 h post inoculation. The results showed that more gene expression differentiation was observed in the compatible interaction induced by T30–4 than that in the incompatible interaction induced by 428–2. The differentially expressed genes induced by 428–2 were significantly enriched in GO terms associated with immune responses and the regulation of defense responses, as well as KEGG pathways related to antioxidant activities, for example, the glutathione metabolism. In contrast, differentially expressed genes induced by T30–4 were significantly enriched in GO terms associated with transcription, translation, and biosynthesis of macromolecules, as well as disease resistance related KEGG pathways, such as phosphoinositide metabolism, phospholipid signaling system, and receptor-mediated endocytosis. Our findings indicated that Huasong 7 responds to incompatible P. infestans isolate through disease resistance and host defense related pathways; while it responds to compatible isolate T30–4 with basic responses. It is also possible that T30–4 contains other unknown Avr genes. In summary, our study could provide an important reference for a better understanding of potato late blight resistance mechanisms of both compatible and incompatible interactions.

Short-term transcriptomic responses of Populus euphratica roots and leaves to drought stress

Populus euphratica Oliv., the Euphrates poplar, is the tallest tree species in the arid desert areas of Northwest China. Investigation of its drought-resistance genes is valuable to increase understanding of drought resistance mechanisms. RNA-seq of leaves and roots under drought simulation by 25% polyethylene glycol-6000 (PEG 6000) were performed at 0, 4, and 12 h. Leaves and roots responded differently to drought via differentially upregulated and downregulated genes; more genes were downregulated than upregulated in both leaves and roots. Additionally, these differentially expressed genes were enriched in different GO terms and KEGG pathways. For example, GO term ‘response to organic substance’ and KEGG pathway ‘nitrogen metabolism’ were enriched for drought-stressed leaves, while GO term ‘cell wall organization or biogenesis’ and KEGG pathway ‘zeatin biosynthesis’ were enriched for drought-stressed roots. The enrichment of the GO term ‘enzyme linked receptor protein signalling pathway’ in both leaf and root drought responses suggests that these tissues may also have similar mechanisms. However, roots under drought stress for four hs responded by activating programed cell death. The KEGG pathway ‘plant hormone signal transduction’ was detected for 4- and 12-h drought-stressed leaves and 12-h drought-stressed roots, suggesting that plant hormone signal transduction plays an important role in both roots and leaves. GO enrichment of upregulated and downregulated genes for leaves and roots reflect differentially regulatory mechanisms of response to drought stress via different biological processes such as the regulation of photosynthesis and auxin signalling pathway in leaves, and the regulation of defence response and water homeostasis in roots. Fifteen candidate genes, including transcription factors, protein kinase, transporter, late embryogenesis abundant protein and mannitol dehydrogenase, were further selected to determine their response to drought using qRT-PCR. The results show that the expression patterns of 13 of the 15 genes correspond to the RNA-seq results. This study provides new insights into the drought response mechanism of P. euphratica and suggests new candidate gene resources.

The Role of a Glucosinolate-Derived Nitrile in Plant Immune Responses.

Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.

A strawberry mitogen-activated protein kinase gene, FaMAPK19, is involved in disease resistance against Botrytis cinerea

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules involved in a wide range of resistance strategies against pathogen attack. However, the function of strawberry (Fragaria × ananassa) MAPKs in resistance against Botrytis cinerea remains unclear. The present study investigated the functional role of FaMAPK19 in the regulation of defense responses in strawberry fruit to B. cinerea. Changes in FaMAPK19 expression levels throughout whole-plant development and phenotypes of strawberry fruit after B. cinerea inoculation exhibited strong relationships with the level of FaMAPK19 transcripts and severity of gray mold. Transient transformation was carried out to overexpress FaMAPK19 and its antisense transcript in strawberry fruits, which were later analyzed upon B. cinerea inoculation. The distinct phenotypes of transformed fruits indicated that overexpression of FaMAPK19 improved disease resistance to B. cinerea, but antisense expression of FaMAPK19 caused a reduction in disease resistance. Disease resistance–related transcription factors were almost all upregulated in fruits where FaMAPK19 was overexpressed. These results suggest that FaMAPK19 plays a pivotal role in defense responses in strawberry fruit to B. cinerea. Moreover, we found that the transcript levels of some hormone-related genes, some structural genes, and a series of ripening-related genes were highly influenced by overexpression and antisense expression of FaMAPK19 in fruits, suggesting that FaMAPK19 might also have functions in fruit development and ripening. We thus hypothesize that the FaMAPK19 gene plays a key role in fruit disease resistance as well as a variety of biological functions, including fruit development and ripening regulation.

Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death.

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid-dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function.

Transcriptional Profiles of Mdwrky33 in Apple Root in Response to Infection by Pythium Ultimum, Abiotic Stresses and Chemical Treatments

Plant resistance responses to pathogen infection involve massive transcriptional reprograming and widespread redirection of cellular pathways to adjust the plant from growth to defense. Transcription factors (TFs) function at the major regulating points of gene expression, and specific TFs are known to play crucial roles in plant defense activation. Molecular defense activation in apple root from infection by oomycete necrotrophic pathogen Pythium ultimum, a primary component in a pathogen complex inciting apple replant disease, has not been investigated in detail. Base on previous transcriptome analyses, members of apple WRKY gene family have been identified as the primary candidates in regulating defense response in apple root. Among them, MdWRKY33, an orthologue of AtWRKY33 in apple genome, demonstrated as a highly-expressed WRKY with genotype-specific induction patterns during P. ultimum infection. The sequence features of MdWRKY33 and its tissue-specific expression, as well as its responses to abiotic and pharmacological treatments, added to the evidence for its functional roles in defense activation in apple root. In response to P. ultimum infection, MdWRKY33 was consistently upregulated in all eight tested apple rootstock genotypes at all timepoints. Between genotypes, the stronger induction patterns at the earlier stage of infection in resistant genotypes suggest its essential roles of contributing to apple root resistance, although plant resistance to necrotrophic pathogens is polygenetic quantitative resistance in nature. Transgenic manipulation of this gene is underway to provide more definitive functional identity in contributing to apple root resistance to P. ultimum infection

Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.